Structure of thermolysin refined at 1.6 Å resolution

Holmes, Michael; Matthews, Brian W.

doi:10.1016/0022-2836(82)90319-9

Cited by 451 publications

(276 citation statements)

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“…For this study of limited proteolysis of a model protein in its TFE-state, the thermophilic enzyme thermolysin (Heinrikson, 1977;Holmes & Matthews, 1982) appeared to be a most suitable proteolytic probe, because of its noteworthy stability under relatively harsh solvent conditions, including aqueous organic solvents (Wayne & Fruton, 1983;Welinder, 1988;Kitaguchi & Klibanov, 1989) and broad substrate specificity (Heinrikson, 1977;Keil, 1982). Thus, it was anticipated that thermolysin would cleave the polypeptide chain of RNase in its TFE-state at sites characterized by the flexibility required for an efficient proteolysis and not by the specificity of the protease.…”

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Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42"C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Thl) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

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Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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“…Native crystals suitable for X-ray diffraction were prepared as described by Holmes and Matthews (1982) and kept in a mother liquor of 0.01 M calcium acetate, 0.01 Tris acetate, and 5% (by volume) dimethyl sulfoxide, pH 6.8. To prepare metalsubstituted enzyme crystals, the mother liquor was gradually replaced (over 1 day) by an equivalent solution that lacked zinc but contained the replacement metal ion (100 mM).…”

Section: Methodsmentioning

confidence: 99%

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1995

Protein Science

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Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, JBiol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution.The structure of the Co2+-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobaltrelative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd2+-and Mn2+-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 1 4 4 . Some of these sidechain movements are similar to adjustments that have been observed previously in association with the "hingebending" motion that is presumed to occur during catalysis by the zinc endoproteases.In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect. (Fig. 1) showed the zinc liganded tetrahedrally by His 142, His 146, Glu 166, and a water molecule, hereafter referred to as Wat 231 (Fig. 2). Further refinement has suggested that, in addition to Wat 23 1, there is also a dipeptide, presumably a product of selfproteolysis, bound to the active site (Holland et al., 1992 (1974, 1976) have shown that removal of zinc from thermolysin yields an inactive apoenzyme and that varying levels of esterase and peptidase activity can be restored upon substitution of the native zinc atom with transition metals. In particular, Zn2+, Co2+, and Mn2+, when added in stoichiometric amounts, restored 100,200, and 10% of the activity of the native enzyme toward furylacyloylglycyl-L-leucyl amide (FAGLA). Furthermore, in concentrations up to 1 mM, these metals retained or restored catalytic activity to some extent. Fe2+ in high molar excess restored about 60% of native activity. Zn2+ in excess of the amount required for catalytic activity actually inhibited the enzyme. The transition metals Mg2+, Cr2+, Ni2+, Cu2+, Mo2+, Pb2+, Hg2+, Cd2+, Nd2+...

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“…The folding and stability properties of fragments of thermolysin, a well-characterized metalloprotease isolated from Bacillus thermoproteolyticus (Titani et al, 1972;Holmes & Matthews, 1982), have been the subject of growing interest in recent years (Vita et al, 1979(Vita et al, , 1984(Vita et al, , 1989Vita & Fontana, 1982;Dalzoppo et al, 1985;Conejero-Lara et al, 1994;Azuaga et al, 1995;Conejero-Lara & Mateo, 1996). This was due to the finding that short amino acid sequences of the protein fold into native-like conformations and undergo cooperative unfolding transitions under the appropiate conditions.…”

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“…Thermolysin consists of a single polypeptide chain of 316 amino acid residues with a functional zinc ion and four calcium ions and has no thiol or disulfide groups. The three-dimensional structure of thermolysin, determined by X-ray, shows that this protein is constituted of two structural domains of equal size (residues 1-157 and 158-316), with the active site located at the interface between them (Holmes & Matthews, 1982). As mentioned above, some fragments of thermolysin, namely, 121-316, 206-316, 228-316, and 255-316, all belonging to the C-terminal domain, were shown by spectroscopic and immunochemical techniques to refold in water into stable native-like structures (Fontana, 1990;Vita et al, 1989).…”

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NMR Solution Structure of the 205−316 C-Terminal Fragment of Thermolysin. An Example of Dimerization Coupled to Partial Unfolding

Conejero‐Lara

González

et al. 1997

Biochemistry

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The solution structure of the C-terminal fragment 205-316 of thermolysin has been determined by 1 H-NMR methods. The fragment forms a dimer in which each subunit has two different regions: the largely disordered N-terminal segment 205-260 and the structurally well-defined segment 261-316. The structured part of each subunit is composed of three helices and is largely coincident with the corresponding region in the solution structure of the dimer formed by the shorter fragment 255-316, which in turn coincides with the crystallographic structure of intact thermolysin. As with the fragment 255-316, the subunit interface is highly hydrophobic and coincides topologically with the one between the segment 255-316 and the rest of the protein in the intact enzyme. A fourth helix (residues 235-246), present in the segment 205-316 of native thermolysin, is mostly disordered in the dimer formed by the fragment 205-316. The location of the fourth helix in the native structure of intact thermolysin does not allow the formation of the dimer interface observed in the solution structure of the fragment 255-316. Under the NMR conditions, dimer formation is energetically more favorable than the dissociated monomers. The latter, based on calorimetric data, was proposed to have partial structure in the region 205-254 as in native thermolysin. Thus, it appears that the assembly of the dimer would require an initial unfolding in the region 205-254 of the monomer.

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Structure of thermolysin refined at 1.6 Å resolution

Cited by 451 publications

References 25 publications

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

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NMR Solution Structure of the 205−316 C-Terminal Fragment of Thermolysin. An Example of Dimerization Coupled to Partial Unfolding

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